PREPARATION OF PLASMID DNA

Confirm the colony has the appropriate phenotype by growth on antibiotic plates, and the plasmid has the appropriate size insert using the "plasmid quick prep" procedure. Freeze a portion of the quick prep culture in L broth and 15% glycerol at -70°C for future inoculations.

Inoculate a 20-50 ml overnight culture (with the appropriate antibiotic). The next day, add this to 1 to 2 liters of L broth with the appropriate antibiotic and grow to a density of about 150 Klett units or A550 = 0.9-1.0. Depending on the volume of the initial inoculum, this usually takes about 3 hours. For pBR plasmids, add chloramphenicol to 100 µg/ml and shake from 4 hours to overnight ( pUC plasmids don't need chloramphenicol amplification). Pellet the bacteria in a centrifuge at 4200 xg for 10 minutes at 4°C in 250 - 500 ml centrifuge bottles in an appropriate rotor (5000 rpm in a Sorvall GS-3 rotor). Resuspend the bacterial pellet in 50-100 ml of solution #1. Centrifuge again at the same speed and discard the supernatant. Freeze the bacterial pellet at least 30 minutes in the -70°C freezer to enhance lysis (overnight or longer periods of freezing work fine).

Preparation of Cleared Lysate
For a 1 to 2 liter culture follow this protocol (double for 3-4 liters). One liter usually yields a few mg of plasmid DNA.

  1. Resuspend the pellet in 50 ml cold 25% sucrose in 50 mM Tris-HCl pH 8 (Solution #2 ). Keep the bacteria on ice while resuspending.
  2. Add 10 ml lysozyme (5 mg/ml in 0.25 M Tris-HCl pH 8 solution #3; invert to dissolve). Swirl gently on ice for 5 minutes.
  3. Add 20 ml 0.25 M (10 ml 0.5M) EDTA (solution #4), swirl by hand a few times to mix, and then incubate on ice for 5 minutes without agitation.
  4. Add at the same time 5 µl of a 10 mg/ml stock of RNAse A and 25 ml of triton Lytic Mix (Solu tion #5). Swirl gently on ice for 15 minutes. (The amount of RNAse is probably an overkill; original protocol: 10 µl of 2 mg/ml stock in 10 mM Tris-HCl, pH 7.4 and 1 mM EDTA, boiled for 30 minutes and stored at -30°C).
  5. Spin for 45 minutes at 4°C at 20K rpm in an SS34 rotor/RC5B centrifuge (or in a J2-21 centrifuge/JA-20 rotor).
  6. Carefully pour and save the upper, less viscous supernatant. Leave the more viscous, clear liquid (which is not always present) and the cloudy, white debris (affectionately known as "snot-balls") in the bottom of the tube. I usually transfer the top supernatant to a 50 ml disposable, conical centrifuge tube, keeping the final volume < 34 ml/tube.
  7. To this cleared lysate add 1/2 volume of PEG (Solution #6) and invert a few times to mix. (If you use the 50 ml tube with 33-34 ml of the cleared lysate per tube, add the PEG solution to the 50 ml mark.) Place at 4°C for a minimum of 2 hours (ove rnight is fine).
Plasmid DNA Isolation
  1. Centrifuge the PEG precipitate at 1800 xg for 5 minutes at RT. When using the Beckman TJ-6 table top centrifuge, spin at 3000 rpm. You should see the precipitate in the bottom of the centrifuge tube. Discard the supernatant. Drain tubes on paper towels for 5 minutes to remove residual liquid.
  2. Dissolve the PEG precipitate in a few ml of solution #7 and transfer to a single container. QS to the approp riate volume with solution #7 for the desired rotor.

    Rotor ml Solution #7 g CsCl ml Eth. Br (5 mg/ml)
    Ti50 6.1 6.2 0.5
    Ti80 7.05 7.5 0.5
    VTi65.1 8.2 8.77 0.5

  3. Dissolve the CsCl (technical grade is fine) in this solution, then add the ethidium bromide (Solution #8) - dim the room lights. When everything is dissolved, adjust the refractive index to 1.390 (from 1.3895 to 1.3905). Use the refractive index calculator on the computer (program name "CSCL"). Fill and balance ultracentrifuge tubes to within 50 mg.
  4. Centrifuge according to the following table.

    Rotor Time (hrs) RPM (1/K) Temp (°C)
    Ti50 48-72 42-44 20
    Ti80 48-72 44 20
    VTi65.1 16 (overnight) 40 20

  5. After centrifugation, two bands should be visible about half-way down the gradient. The lower band is the supercoiled plasmid DNA. Illuminate the centrifuge tube with long wave ultraviolet light. For quick-seal tubes, puncture a hole in the top of the tube with an 18g needle. Put a small piece of scotch tape on one side of the tube below the plasmid band. Being careful not to puncture yourself, side puncture the tube with an 18g needle and syringe going through the tape and up into the plasmid DNA band. Collect the plasmid band. For tubes other than quick-seal tubes, side punctures c an be done, but we typically withdraw the plasmid DNA through the gradient with excellent results. Carefully remove or push aside the lipid layer on top of the gradient as you remove (with a Pasteur pipet) about 3/4 of the CsCl solution above the upper bacterial DNA band. Slowly remove the upper bacterial DNA (once it starts flowing into the pipet, it will continue to flow even if the pipet is lifted above the band). Be very careful to avoid disturbing the lower plasmid band. A small amount of degraded b acterial DNA and nicked plasmid DNA usually remains. Change pipets (plastic or silanized glass). Remove as much of the supercoiled plasmid DNA band using a slow circular motion in as small a volume as possible to avoid contamination of material that bands lower in the gradient.
  6. Transfer the plasmid DNA to a sterile, 30 ml silanized test tube.
  7. Extract the ethidium bromide with an equal volume of CsCl equilibrated isopropanol (solution #9) four times, covering t he top of the tube with cellophane and discarding the upper, pink isopropanol layer. The upper layer should be clear after the final extraction.
  8. Extract the DNA once with an equal volume of TE or STE saturated phenol, being careful to determine whether or not the phases have inverted. Extract the aqueous phase three times with ether. Add two volumes of TE to the DNA and do a routine ethanol precipitation at -30°C overnight (the CsCl will fall out of solution at -70° C).
  9. Spin the ethanol precipitate at 10K rpm for 30 minutes, 4°C and dry the pellet in a vacuum desiccator. Resuspend the DNA in TE (100-200 µl) to a final concentration of about 1-2 mg/ml. Our plasmid preparations yield 500 µg - 4 mg of plasmid DNA per liter culture.
  10. Always run a small sample of the DNA uncut and cut with the appropriate restriction enzyme to be sure you can isolate the insert before proceeding with a preparative digest & gel. Add extra restriction enzyme to efficiently digest supercoiled DNA.

Recipes
L Broth (LB; Luria-Bertani)
10 g tryptone
5 g yeast extract
5 g NaCl
1 L water
Autoclave
Antibiotic Concentrations (stocks are 1000x concentrates of these values).
Ampicillin 100 µg/ml
Chloramphenicol 100 µg/ml
Tetracycline 20 µg/ml

Solution #1: 0.1M NaCl; 50mM Tris-HCl pH 7.8; 10mM EDTA
20 ml 5 M NaCl
50 ml 1 M Tris-HCl, pH 8.0
20 ml 0.5 M EDTA, pH 8.0
Use HCl to pH to 7.8 (takes approximately 1 ml of concentrated acid).
QS to 1 liter with water and autoclave.
Solution #2: 25% Sucrose in 50 mM Tris-HCl pH 8.0
25 ml 1 M Tris-HCl, pH 8.0
125 g sucrose
Adjust pH with NaOH. QS to 500 ml with wa ter and autoclave.
Solution #3: 0.25 M Tris-HCl pH 8.0
For 200 ml:
50 ml 1 M Tris-HCl, pH 8.0
Adjust pH to 8.0. QS to 200 ml with water and autoclave.
Solution # 4: 0.25 M EDTA, pH 8.0
For 250 ml:
125 ml 0.5 M EDTA pH 8.0
pH with NaOH to 8.0. QS to 250 ml with water and autoclave.
Solution #5: 0.3% Triton X-100 in 0.15M T ris-HCl pH 8.0, 0.18 M EDTA
For 250 ml:
37.5 ml 1 M Tris-HCl pH 8.0
90 ml 0.5 M EDTA pH 8.0
7.5 ml 10% Triton-X 100 (v/v)
Adjust pH to 8.0. QS to 250 ml with water and autoclave.
Solution #6: 30% PEG, 1.5M NaCl
150 ml 5 M NaCl
150 g PEG (mw: 6000 or 8000)
QS to about 500 ml with water and autoclave to dissolve the PEG.
After autoclaving. QS as needed with autoclaved H2O to 500 ml.
Solution #7: 50mM Tris-HCl pH 8.0, 50mM NaCl, 5mM EDTA
For 200 ml:
10 ml 1 M Tris-HCl, pH 8.0
2 ml 5 M NaCl
2 ml 0.5 M EDTA, pH 8.0
QS to 200 ml and autoclave.
Solution #8: Ethidium Bromide Solution (5 mg/ml)
For 100 ml:
Dissolve 0.5 g of ethidium bromide with constant stirring in autoclaved water in a sterile brown glass (or foil wrapped) bott le. This may take overnight to dissolve.
Solution #9: Isopropanol-equilibrated Cesium Chloride Solution
Equilibrate an equal volume of isopropanol with 1 g/ml of CsCl in 1x SSPE.

20X SSPE (3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA, pH 7.0)
350.7 g NaCl
55.2 g NaH2PO4(H20) (--or-- 48 g anhydrous NaH2PO4, mw = 120.0)
14.89 g EDTA
Dissolve in boiling water, cool to room temperature, & adjust pH to 7.0 with NaOH. QS to 2 liters with water and autoclave.
TE (10 mM Tris-HCl pH 7.8; 1 mM EDTA)
For 600 ml solution:
0.727 g Tris
1.2 ml of 0.5M EDTA
pH t o 7.8 with approximately 7 drops of concentrated HCl with a pasteur pipet (ACID - wear rubber gloves), or about 4 ml of 1 M HCl. QS to 600 ml with water and autoclave.
Return to Protocols: Table of Contents
Return to the Frank Lab Home Page
Copyright 1989, 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Preparation of Plasmid DNA. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/plasmids.html). 1997. Oklahoma City. Revision Date: October 2, 1997."